Journal of Industrial and Engineering Chemistry 47 (2017) 272280

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Biosurfactant production by the hydrocarbon-degrading bacteria

(HDB) Serratia marcescens: Optimization using central composite
design (CCD)
Asia Fadhile Almansoorya,e, Hassimi Abu Hasanb,f,* , Mushrifah Idrisa,c ,
Siti Rozaimah Sheikh Abdullahb,c , Nurina Anuarb , El Mubarak Musa Tibind
a
School of Environmental and Natural Resource Sciences, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
b
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi,
Selangor, Malaysia
c
Tasik Chini Research Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
d
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
e
Department of Biology, Science Collage, Basrah University, Basrah, Iraq
f
Research Centre for Sustainable Process Technology (CESPRO), Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM
Bangi, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 3 May 2016 Based on the nutritional and physiological requirements of a native hydrocarbon-degrading bacterium
Received in revised form 15 November 2016 (HDB), Serratia marcescens, an investigation on the potential biosurfactant production by this bacterial
Accepted 25 November 2016 species was conducted. The effects of different carbon sources, nitrogen sources, salinity, pH,
Available online 5 December 2016 temperature, and agitation speeds were extensively studied. The optimal conditions of biosurfactant
production and surface tension were determined using central composite design (CCD) by setting
Keywords: glycerol, peptone and ammonium sulphate (NH4)2SO4 in a range of 37%, 26 g/L and 37 g/L,
Biosurfactant respectively. The results showed that biosurfactant was highly produced at pH 8, a temperature of 30
C, a
Serratia marcescens
salinity of 1% and a speed of 200 rpm after 5 days (120 h) of incubation. The optimal conditions were
Biosurfactant-producing bacteria
obtained at 5% glycerol, 4 g/L peptone and 5 g/L (NH4)2SO4 with a maximum biosurfactant production of
Response surface methodology
Hydrocarbon-degrading bacteria 1.42 g/L and a minimum surface tension of 28.4 mN/m. Thus, the HDB S. marcescens shows good potential
as a biosurfactant-producing bacterium to be used in any environmental application and as an alternative
to chemical surfactants.
2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
reserved.

Introduction industrial systems where temperature extremes are integral

Biosurfactants are stable even under extreme conditions in a
wide range of pH, temperature and salinity. Biosurfactant
production is affected by various factors that depend on the
isolate, the medium (carbon, nitrogen source and salinity) and
operating conditions (pH, temperature and agitation speed). These
factors inuence the amount and type of biosurfactant produced
[1]. Biosurfactants might be useful in extreme environments in
* Corresponding author at: Department of Chemical and Process Engineering,
Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia. Fax: +60 3 89118345.
E-mail addresses: simiabuhasan@gmail.com, hassimi@ukm.edu.my
(H. Abu Hasan), rozaimah@ukm.edu.my (S.R. Sheikh Abdullah).
elements [2]. The other advantages of biosurfactants are their
selectivity and specic activity at extreme temperatures, pH and
salinity [2].
Different types of carbon and nitrogen sources have been used
for biosurfactant production [35]. Diesel, gasoline, crude oil,
glucose, sucrose, and glycerol are good substrates. Meanwhile,
peptone, yeast extract, ammonium sulfate ((NH4)2SO4), and
ammonium nitrate (NH4NO3) can be used as nitrogen sources.
The nature of the carbon substrate affects the quality and quantity
of biosurfactant produced [6]. By limiting the nitrogen source, the
biosurfactant production can be increased, whereas a supplemen-
tation of yeast extract (25%) and NaNO3 (10%) results in little
increase in biosurfactant yield [7]. Previous study found that the
nitrogen sources including yeast extract, urea, peptone and sodium
nitrate had comparable inuence on biosurfactant production [8].

http://dx.doi.org/10.1016/j.jiec.2016.11.043
1226-086X/ 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280
Although there are information on the effect of carbon and
nitrogen sources on biosurfactant production, but the interaction
inuence of these nutrients has not been extensively investigated.
The important characteristics of most organisms are their
strong dependence on pH for cell growth. The effect of pH on the
biosurfactant produced by Pseudomonas aeruginosa was studied
[9], who reported that the yield of pure biosurfactant increases
with an increase in pH from 6 to 10; optimal productions are
achieved at a pH of 9. Agitation is also an important factor that
inuences the biosurfactant production. It links to the physiologi-
cal function of the microbial emulsier, where the production of
bioemulsiers enhances the solubilization of water-insoluble
substrates and consequently facilitates nutrient transport to the
microorganisms [10].
Biosurfactant by-products of the Serratia bacterium are used as
tracers to degrade hydrocarbon in eld experiments [11]. The
biodegradation of hydrocarbons by natural populations of micro-
organisms producing biosurfactant occurs via surface active
agents, which are categorized by their chemical composition
and microbial origin. The biosurfactant is characterized to contain
glycolipids, lipopeptides, polysaccharideprotein complexes, pro-
tein-like substances, lipopolysaccharides and phospholipids [12].
One of the most important characteristics of biosurfactant
produced is its ability to emulsify hydrocarbons in solution by
producing surface-active agents that can cause dispersion of
hydrocarbons in water emulsions into microdroplets or micelles
[13].
The aim of this study was to optimize the biosurfactant
production by the hydrocarbon-degrading bacteria (HDB),
Serratia marcescens under optimal carbon and nitrogen sources
using central composite design (CCD). The HDB S. marcescens
was isolated from a hydrocarbon-contaminated soil in Melaka,
Malaysia. From our previous investigation [14], HDB S. marces-
cens was an efcient bacteria in producing biosurfactant
compared to other 28 isolates. Before optimization, the effects
of carbon, nitrogen, pH, agitation speed, temperature and
salinity were extensively investigated using the one variable
at a time (OVAT) method. Moreover, the effect of the interaction
of the carbon and nitrogen sources on the biosurfactant
production was also studied.
Experimental
Cultivation and medium composition for the HDB S. marcescens in
producing biosurfactant
The HDB S. marcescens strain was isolated from soil contami-
nated by hydrocarbons in Melaka, Malaysia. A mineral salt medium
(MSM) was used as the culture medium for the production of
biosurfactant by the HDB S. marcescens. The medium contained
1.2 g/L KH2PO4, 1.8 g/L K2HPO4, 4.0 g/L NH4Cl, 0.2 g/L MgSO47H2O,
0.01 g/L FeSO47H2O, 0.1 g/L NaCl and 1 mL trace elements. The
composition of the trace elements was 0.1 g/L MnSO4H2O, 0.025 g/
L CuCl2, 0.025 g/L (NH4)6Mo7O244H2O, 0.025 g/L Co(NO9)26H2O,
0.025 g/L ZnCl and 0.01 g/L NH4NO3. The pH of the MSM medium
was maintained between 7.0 and 7.2 using 1 mol/L sodium
hydroxide (NaOH) and hydrochloric acid (HCl).
Effects of carbon and nitrogen sources, salinity levels and operational
parameters on biosurfactant production
The production of biosurfactant by the HDB S. marcescens was
conducted using a 500 mL Erlenmeyer ask containing 100 mL of
culture medium. The production was investigated under the effect
of carbon and nitrogen sources, salinity levels and operational
parameters (pH, cultivation time, temperature, agitation speed).
273
Initially, different pHs (pH 5.0, 6.0, 7.0, 8.0 and 9.0) were tested. For
cultivation time, the experiment was designed for 168 h of
sampling with interval sampling times of 24, 48, 72, 96, 120 and
168 h. Meanwhile, the temperature and agitation speed were
varied at 20, 25, 30, 35 and 40
C and 100, 125, 150, 180 and
200 rpm, respectively.
The effect of the carbon source on the biosurfactant production
was studied by growing the HDB S. marcescens in culture media
with different carbon sources (5%) added such as glucose, fructose,
glycerol, olive oil and sucrose. Selecting the best carbon source to
produce biosurfactants, different nitrogen sources [peptone, yeast
extract, (NH4)2SO4 and a mix of peptone with yeast extract,
peptone with (NH4)2SO4 and, (NH4)2SO4 with yeast extract] were
explored. Moreover, the effect of salinity was investigated by
adding different concentrations of NaCl (15%) to the culture
media. All the factors studied were conducted in triplicate in order
to ensure the precision of the obtained data.
Optimization of carbon and nitrogen sources for biosurfactant
production by the HDB S. marcescens
A design of the experiment method using central composite
design (CCD) packaged in Design Expert1 6.0.10 (USA) was applied
to optimize the production of biosurfactant. Three independent
factors, such as the glycerol (x1: 37%), peptone (x2: 26 g/L) and
(NH4)2SO4 (x3: 37 g/L) concentrations, that had been identied
earlier to produce the highest biosurfactant were further exam-
ined. The total experiment simulated by the Design Expert1 was 20
runs comprising 14 runs coupled with 6 center point runs of the
design to assess the magnitude of error that occurs randomly. The
dependent responses were biosurfactant production (y1) and
surface tension activity (y2).
The results were analyzed to develop an appropriate model
for these responses and factors. An analysis of variance (ANOVA)
was performed to obtain the signicant effect of the mani-
pulated independent factors on biosurfactant production and
surface tension activity through the F-values and p-values. The
quality of the t of the polynomial model was expressed by the
determination coefcient R2 and adjusted R2 (R2adj). A module in
Design Expert1 software searched for a combination of factor
levels that would simultaneously satisfy the requirements placed
on each of the responses and factors. The objective function of the
optimization of biosurfactant production was to maximize the
production of biosurfactant and minimize the surface tension by
optimizing the concentrations of glycerol, peptone and
(NH4)2SO4. The mathematical relationships between the
responses and independent factors can be represented by
quadratic models.
Extraction of the crude biosurfactant
After incubation of the HDB S. marcescens in a 100 mL culture
medium, the S. marcescens cells were removed by rst-stage
centrifugation at 4
C and a speed of 8000 rpm for 15 min. The cell-
free supernatant was harvested and adjusted to pH 2 with 1 N HCl.
The biosurfactant was collected via second-stage centrifugation at
4
C and a speed of 12,000 rpm for 15 min. The dry pellet was
lyophilized to produce the biosurfactant in the form of a white
sediment. This procedure was a modication of a method from a
previous study [15]. The dry weight of the produced biosurfactant
was calculated using Eq. (1) as follows:
B B1  B0 1
where B is the mass of the biosurfactant, B0 is the mass of the
empty plate and B1 is mass of the plate containing biosurfactant
after drying at 105
C for 1 h.
274 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280
Measurement of surface tension
The supernatant after second-stage centrifugation at
12,000 rpm for 15 min was used to measure the surface tension
activity using a du Nouy ring-type tensiometer (KSV-sigma 703D,
Finland). The bacterial cell growth was monitored by measuring
the dry cell weight, which was determined by rst-stage
centrifugation of the incubated culture medium. The cell pellet
was washed with distilled water twice and dried by heating at
50
C until a constant weight was attained [16].
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) was used to
elucidate the chemical functional groups in crude biosurfactants.
About 5 mg biosurfactant samples were milled with 80 mg of
potassium bromide (KBr) to form a very ne powder. This powder
was then compressed into a thin pellet which could be analyzed by
the FT-IR system (Perkin Elmer Spectrum BX). The spectral
measurements were carried out in absorbance mode.
Results and discussion
Effect of pH and cultivation time
Environmental conditions play a key role in the growth of
microorganisms, and biosurfactant production is affected both
qualitatively and quantitatively. As shown in Fig. 1(a), the
production of biosurfactant at a pH range of 59 was 0.30
0.85 g/L. The highest production of biosurfactant by S. marcescens
was achieved at pH 8 with 0.85 g/L production and 31.9 mN/m
surface tension. At pH 5 and 11, the biosurfactant production was
0.30 and 0.74 g/L, respectively. The production of biosurfactant by
Fig. 1. Effect of (a) pH and (b) cultivation time on biosurfactant production and
surface tension.
S. marcescens strain DT-IP was also reported as being achieved at
pH 8 [17]. Moreover, the biosurfactant production by Bacillus
mycoides and the reduction in surface tension were highest under
the optimal growth conditions of pH 78 [18].
As shown in Fig. 1(b), at a cultivation time of 24168 h, the
biosurfactant production and surface tension were in the range of
0.361.36 g/L and 23.430.9 mN/m, respectively. The highest yield
of 1.36 g/L was achieved at 120 h (5 days) of cultivation and a
20.9 mN/m surface tension. A biosurfactant is a secondary
metabolite that increases during the late log-phase of growth
[19]. The production of biosurfactant reached up to 62% after 8
9 days of cultivation [12]. By using S. marcescens DT-IP, the
biosurfactant production increased with cultivation time, where
the yield started in the early stationary phase and reached a
maximum in 8 days [20].
Effect of carbon and nitrogen sources
A fundamental aspect in the improvement of biosurfactant
productivity is the carbon source contained in the medium, as it
can encourage the production of biosurfactant. As shown in
Fig. 2(a), the best carbon source for producing biosurfactant was
glycerol with a concentration of 1.05 g/L for biosurfactant
production and a low surface tension value of 30.4 mN/m. A
comparison between the ve carbon sources in producing
biosurfactant efciently provided the following order: glycerol >
olive oil > glucose > sucrose > fructose. It was found that S. mar-
cescens NSK-1 produced high biosurfactants and low surface
tension when cultivated in glycerol as the sole carbon source [21].
The decrease in surface tension indicates the production of wetting
agent compounds (biosurfactant) by the bacterial strain. The
results of this study indicate that biosurfactant production is
growth-associated because a parallel relationship existed between
S. marcescens growth and biosurfactant production, as shown by
the reduction in surface tension and increase in biosurfactant yield.
Fig. 2. Effect of (a) carbon sources and (b) nitrogen sources on biosurfactant
production and surface tension.
 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280 275

Nitrogen is necessary to complete the metabolic pathway of

biosurfactant production. Fig. 2(b) shows that (NH4)2SO4 and
peptone were the best combination in producing the biosurfactant
with a yield of 1.33 g/L (and surface tension 29.9 mN/m), followed
by a combination of yeast extract + peptone (1.23 g/L), peptone
(0.88 g/L), a combination of (NH4)2SO4 + yeast extract (0.81 g/L),
(NH4)2SO4 (0.80 g/L) and yeast extract (0.75 g/L). The biosurfactant
production by Pseudomonas sp. BPB7 and Serratia sp.
BPB13 increased when (NH4)2SO4 was added to the culture
medium [11]. This increase occurred as a result of the direct intake
of amino acids as precursors for surfactant biosynthesis, thus
improving the biosurfactant yield. (NH4)2SO4 as a nitrogen source
plays a signicant role in biosynthesis and extracellular secretion
[12]. It is easily utilized by S. marcescens for cell metabolism
because the solubility of this salt in water is higher than that of
other nitrogen sources. The medium composition, such as carbon
and nitrogen sources and other growth factors, is strongly
inuenced by cell growth and the accumulation of metabolic
products; thus, the optimization of these parameters can improve
the bacterial efciency [18].

Effect of temperature, different RPMs and salinity

As shown in Fig. 3(a), the cultivation of S. marcescens under a

temperature of 30
C produced more biosurfactant (1.35 g/L) and
less surface tension (27.8 mN/m) than other temperature
conditions (20, 25, 35 and 40
C). A previous investigation found
that S. marcescens can produce red pigment prodigiosin and the
biosurfactant serrawettin at 30
C but not at 37
C [22]. A
temperature of 30
C has also been reported as the most favorable
condition for Candida species, viz., Candida sp. SY16 and Candida
bombicola, in yielding biosurfactant [23]. Fig. 3(b) shows the effects
of different RPMs on biosurfactant production and surface tension.
The highest production of biosurfactant (1.33 g/L) and the lowest
surface tension (27.8 mN/m) were achieved at an agitation speed of
200 rpm. Meanwhile, the lowest production of biosurfactant
(0.80 g/L) and the highest surface tension (36.7 mN/m) occurred
at an agitation speed of 100 rpm.
The salinity was found to be a critical parameter in the
production of biosurfactant; in the absence of salt, the production
and growth were very low. As found in this study (Fig. 3(c)),
increasing the salinity from 1 to 5% inhibited the production of Fig. 3. Effect of (a) temperature, (b) different RPMs and (c) NaCl concentration on
the biosurfactant production and surface tension.
biosurfactant by S. marcescens and increased the surface tension.
The most suitable salinity condition in producing biosurfactant
with a combination of 5.0% glycerol, 4 g/L peptone and 5 g/L
efciently was 1%, with a biosurfactant yield of 1.57 g/L and a
NH4(SO4)2. A quadratic model was built to t the results of all
surface tension of 28.4 mN/m. It was reported that the
simulated runs. The quadratic model obtained is given as Eqs. (2)
S. marcescens strain NSK-1 showed tolerance for up to 12% NaCl
and (3).
[21], suggesting that S. marcescens can be potentially applied over a
wide range of high-salt environments in yielding biosurfactant. y1 1:42  0:067x1 0:029x2  0:100x3  0:28x21  0:27x22
2
Evaluation of simulated run by CCD 0:18x 3  0:063x1 x2  0:023x1 x2  0:038x2 x3 2

After conducting the one variable at a time (OVAT) method for

the operational parameters (pH, cultivation time, temperature,
agitation speed, salinity), CCD was utilized as a statistical design to
determine the relationship between the factors (concentration of
glycerol, peptone and ammonia) and the response (biosurfactant
production and surface tension). Two responses (y1 and y2) were
evaluated in the simulated run by CCD, as summarized in Table 1.
All the actual values were obtained at the optimized operational
conditions (pH 8, cultivation time of 120 h, temperature of 30
C,
agitation speed of 200 rpm, and 1% salinity). Throughout the runs,
run number 17, with the conditions of 7.0% glycerol, 2 g/L peptone
and 3 g/L NH4(SO4)2, yielded the highest biosurfactant production
(1.5 g/L) with low surface tension (27.8 mN/m). The lowest
production of biosurfactant was obtained under run number 8,
y2 28:52 0:062x1  0:96x2  1:16x3 3:69x21 1:55x22
1:99x 23  1:55 x1 x2 0:50x1 x3  2:85x2 x3 3
The models were used to determine the biosurfactant produc-
tion and surface tension under optimal conditions. The ANOVA
results of biosurfactant production and surface tension are
presented in Table 2. The model signicantly (p < 0.05) represents
the relationship between the responses (biosurfactant production
and surface tension) and the signicant input variables. The
F-values for both models were 43.7 and 3.15 for biosurfactant
production and surface tension, respectively. An adequate level of
precision measures the signal-to-noise ratio, and a ratio greater
than 4 is desirable. In the quadratic models of the biosurfactant
276 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280

Table 1
Experimental layout designed by Design Expert and the corresponding experimental and predicted values of the responses.

Run x1 (v/v)% x2 (g/L) x3 (g/L) y1 y2

Actual value Predicted value Error (%) Actual value Predicted value Error (%)
1 8.36 4.00 5.00 0.74 0.70 5.7 32.40 33.91 4.4
2 5.00 4.00 8.36 0.75 1.3 1 35.20 36.14 2.6
3 3.00 6.00 3.00 0.97 0.96 1 36.70 40.80 10.0
4 5.00 4.00 5.00 0.72 0.75 4 35.60 36.82 3.2
5 5.00 0.64 5.00 0.71 0.62 14.5 33.70 36.29 6.9
6 5.00 4.00 1.64 0.62 0.57 8.7 40.80 40.51 0.7
7 1.64 4.00 5.00 0.78 0.73 6.8 28.90 31.77 8.8
8 5.00 4.00 5.00 0.45 0.43 4.6 27.50 29.80 7.7
9 5.00 4.00 5.00 0.66 0.74 10.8 43.60 38.85 12.3
10 3.00 6.00 7.00 0.51 0.51 0 39.70 39.06 1.6
11 3.00 2.00 3.00 0.53 0.61 13.1 35.50 34.52 2.8
12 3.00 2.00 7.00 0.71 0.71 0 35.70 31.30 14
13 7.00 6.00 3.00 1.09 1.08 0.9 38.90 36.11 7.7
14 7.00 2.00 7.00 0.65 0.74 12.1 34.80 32.20 8.07
15 5.00 4.00 5.00 1.40 1.42 1.4 26.80 28.52 5.9
16 5.00 4.00 5.00 1.42 1.42 0 29.50 28.52 3.5
17 7.00 2.00 3.00 1.50 1.42 5.6 27.80 28.52 2.4
18 7.00 6.00 7.00 1.30 1.42 8.4 27.60 28.52 3.15
19 5.00 7.36 5.00 1.50 1.42 5.6 32.70 28.52 14.7
20 5.00 4.00 5.00 1.41 1.42 0.7 25.78 28.52 9.8

production and surface tension obtained, the ratios of 17.60
(biosurfactant production) and 4.82 (surface tension) indicate an
adequate signal for the models to be used to navigate the design
space.
The obtained lack of t F-values of 1.28 and 3.31 in Eqs. (1) and
(2) indicate the insignicance of the pure errors for the
biosurfactant production and surface tension, respectively. The
normal % probability versus studentized residuals graphs for the
responses of y1 and y2 yielded fairly straight lines, implying a
normal distribution of the data. Moreover, the outlier-t values lie
below the interval of 3.50, indicating that the approximation of
the tted model to the response surface was fairly good with no
data recording error [24]. The quadratic R2 values were 0.975 and
0.739 for biosurfactant production and surface tension, respec-
tively. According to the software and specied section of
optimization, the statistical condence of the model was 95%,
which is appropriate. The R2 value indicates how much of the
variability in the data was accounted for by the model, while the
adjusted R2 modies the R2 value by taking into account the
number of covariates or predictors in the model. This R2 coefcient
ensures a satisfactory adjustment of the quadratic model to the
experimental data [18]. The adjusted R2 values were 0.9529 and

Table 2
ANOVA analysis of the quadratic model for biosurfactant production and surface tension.

Source Sum of squares Degrees of freedom Mean square F-value Prob > F
Biosurfactant production
Model 2.49 9 0.28 43.72 0.0001s
x1 0.061 1 0.061 9.64 0.1110ns
x2 0.012 1 0.012 1.88 0.2004ns
x3 0.14 1 0.14 21.43 0.0009s
x12 1.14 1 1.14 180.10 0.0001s
x22 1.04 1 1.04 164.59 0.0001s
x32 0.47 1 0.47 74.10 0.0001s
x1x2 0.031 1 0.031 4.95 0.0504s
x1x3 4.050E-003 1 4.050E-003 0.64 0.4420ns
x2x3 0.011 1 0.011 1.78 0.2117ns
Residual 0.063 10 6.319E-003
Lack of t 0.036 5 7.101E-003 1.28 0.3957ns
Pure error 0.028 5 5.537E-003
R2 = 0.9752, R2adj = 0.9529, Adeq precision = 17.60

Surface tension
Model 367.95 9 40.88 3.15 0.0442s
x1 0.052 1 0.052 3.989E-003 0.9509ns
x2 12.50 1 12.50 0.96 0.3497ns
x3 18.50 1 18.50 1.43 0.2601ns
x12 196.33 1 196.33 15.12 0.0030s
x22 34.71 1 34.71 2.67 0.1331ns
x32 57.30 1 57.30 4.41 0.0620ns
x1x2 19.22 1 19.22 1.48 0.2561ns
x1x3 2.00 1 2.00 0.15 0.7029ns
x2x3 64.98 1 64.98 5.01 0.0492s
Residual 129.82 10 129.82
Lack of t 99.71 5 99.71 3.31 0.1074ns
Pure error 30.12 5 30.12
R2 = 0.7392, R2adj = 0.5045, Adeq precision = 4.82

ns indicates no signicant difference at p > 0.05.

s indicates a signicant difference at p < 0.05.
 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280
0.5405 for the two models y1 and y2, respectively; these values
indicate the signicance of the models [25].
Optimization of biosurfactant production
The goal in the optimization of biosurfactant production was to
obtain the maximum production of biosurfactant and minimum
surface tension by setting the concentrations of glycerol, peptone
and (NH4)2SO4 in the range of the study conditions. Through the
optimization, the individual desirability was combined into a
single number and then searched to maximize the model (Eqs. (2)
and (3)) functions. The optimized condition of each factor was
4.87% (glycerol), 4.24 g/L (peptone) and 4.98 g/L ((NH4)2SO4) with a
desirability of 0.888, as depicted in Fig. 4. The biosurfactant
production at these conditions was 1.42 g/L with a surface tension
of 28.4 mN/m. To validate the obtained optimal conditions, a
validation experiment was executed to conrm the produced
biosurfactant and surface tension activity. In this conrmatory run,
the errors between the results obtained from the DOE model and
validation experiment were 5.3% and 5.9% for biosurfactant
production and surface tension, respectively.
Effect of the interaction of each factor on biosurfactant production and
surface tension
The three-dimensional (3D) response surface plots (Figs. 5 and
6) illustrate how biosurfactant (y1) and surface tension (y2) relate
to the factors of glycerol (x1), peptone (x2) and (NH4)2SO4 (x3)
through the quadratic models in Eqs. (2) and (3). Figs. 5 (a) and 6
(a) show the 3D surface plot of the interaction effect of glycerol (x1)
and peptone (x2) on biosurfactant production and surface tension
at the optimal (NH4)2SO4 concentration of 5 g/L. It can be observed
that glycerol within the range of 3.007.00% (v/v) led to steady
biosurfactant production and surface tension. Peptone (x2) gave an
increased biosurfactant yield within 45 g/L. The interaction of
glycerol and peptone signicantly affected the biosurfactant
production (p < 0.05) but not the surface tension activity. It is
Fig. 4. Optimal condition of biosurfactant production.
277
generally believed that increasing peptone to 4 g/L encourages
higher biosurfactant production and lower surface tension.
Moreover, a higher biosurfactant yield was achieved using 3.5%
glycerol as the substrate [20]. The cultivation of P. aeruginosa in
glycerol reduced the surface tension of the culture medium and
yielded a biosurfactant production of 1.06 g/L [26].
Figs. 5 (b) and 6 (b) show the interaction effect of glycerol (x1)
and (NH4)2SO4 (x3) on biosurfactant production and surface
tension at the optimal peptone concentration of 4 g/L. The
biosurfactant production signicantly increased (p < 0.05) when
(NH4)2SO4 was increased from 3 to 5 g/L. However, the interaction
of glycerol and (NH4)2SO4 did not signicantly affect the
biosurfactant production, although it signicantly reduced the
surface tension activity. It was favorable when using glycerol and
(NH4)2SO4 at a C:N ratio of 5.5 for biosurfactant production by S.
marcescens [27]. Using (NH4)2SO4 as a nitrogen source in
cultivation and production medium, it was found that the
biosurfactant yield produced by Candida utilis was 12.52 g/L at
the lowest surface tension activity of 43.19  1.98 mN/m [28].
Figs. 5 (c) and 6 (c) show the interaction effect of peptone (x2)
and (NH4)2SO4 (x3) on biosurfactant production and surface
tension at an optimal glycerol level of 5.0%. The statistical analysis
showed that the interaction was insignicant for biosurfactant
production, but it signicantly affected the surface tension
reduction (p < 0.05). As shown in the gure, by increasing the
(NH4)2SO4 content, the biosurfactant production increased and the
surface tension decreased. Among these two nitrogen sources, the
combination of peptone and (NH4)2SO4 at 4 and 5 g/L, respectively,
was the best concentration for the reduction of the surface tension
(28.4 mN/m). Nitrogen sources have been shown to play an
important role in biosurfactant production, and the nature of the
nitrogen source also has an effect on biosurfactant production.
Characterization of biosurfactant produced by the HDB S. marcescens
The chemical functional groups of the biosurfactant produced
by the HDB S. marcescens were investigated using the FTIR
278 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280

Fig. 5. Response surface for biosurfactant production as a function of the interaction between factors: (a) glycerol and peptone, (b) glycerol and (NH4)2SO4, and (c) peptone
and (NH4)2SO4.
 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280 279

Fig. 6. Response surface for surface tension as a function of the interaction between factors: (a) glycerol and peptone, (b) glycerol and (NH4)2SO4, and (c) peptone and
(NH4)2SO4.
280 A. Fadhile Almansoory et al. / Journal of Industrial and Engineering Chemistry 47 (2017) 272280

Fig. 7. Infrared spectrum of puried biosurfactant produced by the HDB Serratia marcescens.

technique. The infrared spectrum of the FTIR analysis is shown in Research Centre and Ministry of Education Malaysia (Grant nos:
Fig. 7. The wave number for each specic band includes a strong FRGS 32-1310063 and FRGS/1/2014/TK05/UKM/02/1) for support-
band at 3534 cm1, indicating the presence of a hydroxyl group. ing this research project. We also acknowledge the Iraqi Ministry of
Apart from functional groups, the acetyl presence in the Higher Education and Scientic Research for providing a doctoral
biosurfactant also provides hydrophilicity to enhance the emulsi- scholarship for the rst author.
fying activity. The peak at 2929 cm1 was associated with the
stretching vibration of the C H bond of the constituent sugar References
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Malaysia (Grant nos: DIP-2014-020 and DIP-2016-030), Tasik Chini